The present invention relates to immunotherapy, particularly to immunotherapy using cytotoxic T lymphocytes (CTL), and more particularly to adoptive immunotherapy.
There is evidence that anti-tumour CTL and anti-virus CTL play an important role in vivo. Tumour-reactive CTL have been shown to mediate tumour regression in animal models (1) and in man (2). Similarly, recent studies suggest that HIV-specific CTL may limit HIV virus load in vivo (3).
There is much interest in using in vitro generated CTL for adoptive immunotherapy of cancer. The potential importance of in vitro generated CTL is suggested in experiments with adenovirus transformed murine tumour cells (1). Nude mice were injected with tumour cells and large tumours were allowed to form. Tumour regression was observed when these mice were treated with CTL specific for the transforming E1A protein expressed in the tumour cells. Similarly, when in vitro generated CTL specific for gp100 were given to a melanoma patient tumour regression was observed (2). Thus, it is believed that adoptive transfer of T lymphocytes with defined specificity represents a promising therapy for cancer patients. Similarly, adoptively transferred CTL specific for cytomegalovirus seem to suppress CMV infection in patients who underwent bone marrow transplantation (4).
WO 93/17095 describes a method of producing, loading and using MHC class I molecules to specifically activate CTL derived from a patient in vitro and then returning the patient""s activated CTL in a form of treatment. WO 93/17095 specifically teaches that it is the patients own CTL that should be used to treat the patient.
Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658 describes the production of functional three-domain single-chain T-cell receptors.
Moritz et al (1994) Proc. Natl. Acad. Sci. USA 91, 4318-4322 describes CTL with a grafted recognition specificity for ERBB2-expressing tumour cells.
Roberts et al (1994) Blood 84, 2878-2889 describes the targeting of HIV-infected cells by CD8+ T lymphocytes armed with xe2x80x9cuniversalxe2x80x9d (chimaeric) T-cell receptors.
U.S. Pat. No. 5,359,046 describes xe2x80x9cuniversalxe2x80x9d (chimaeric) T-cell receptors.
Faber et al (1992) J. Exp. Med. 176, 1283-1289 describes the generation of leukaemia-reactive CTL clones from genotypically HLA-identical bone marrow donor of a patient with leukaemia. In prior art allogeneic bone marrow transplantations the material comes directly from a healthy donor, and so is a mixed population and is not cloned.
A major rate limiting step of current adoptive immunotherapy is that it is patient-specific and dependent upon the isolation and in vitro expansion of specific CTL from the patient""s own T lymphocyte pool. Thus, for each patient elaborate time-consuming and expensive in vitro work is required to generate sufficient numbers of specific CTL. Furthermore, in some patients the immune system may be severely suppressed, and it may be impossible to isolate specific CTL.
The present invention is aimed at overcoming these limitations and providing more efficient and potentially more effective adoptive immunotherapy with cytotoxic T lymphocytes (CTL) of patients, particularly cancer patients.
A first aspect of the invention provides a method of treating a patient with a disease wherein the patient contains diseased cells which cells contain, or are associated with, an abnormal molecule or an abnormally elevated amount of a molecule and which cells are capable of presenting at least part of said molecule on their surface by an HLA class I (or equivalent) molecule, the method comprising administering to the patient a therapeutically effective amount of cytotoxic T lymphocytes (CTL) which recognise at least part of said molecule when presented by an HLA class I (or equivalent) molecule on the surface of a cell characterised in that the cytotoxic T lymphocytes are not derived from the patient with a disease.
Thus the present invention overcomes the previous problems by, for example, generating CTL from, preferably, healthy individuals against selected peptides presented by the patient""s HLA class I molecules. These CTL may be allo-restricted if the CTL donor does not express the class I molecule that presents the CTL recognised peptides, or they may be self MHC(HLA)-restricted if the CTL donor expresses the class I molecule that presents the CTL recognised peptides.
The CTL for administering to the patient are conveniently made using the method of the third aspect of the invention as described below.
By xe2x80x9cHLA class I (or equivalent molecule)xe2x80x9d we mean a HLA class I protein or any protein which is equivalent to a human HLA class I molecule from any other animal, particularly a vertebrate and especially a mammal. For example it is well known that in the mouse the MHC class I proteins are similar in structure to, and fulfill a similar role to, the human HLA class I proteins. Equivalent proteins to human HLA class I molecules can be readily identified in other mammalian species by a person skilled in the art, particularly using molecular biological methods.
By xe2x80x9cat least part of said moleculexe2x80x9d we include any fragment of said molecule that can be presented on the surface of a cell by an HLA class I (or equivalent) molecule.
By xe2x80x9can abnormally elevated amount of a moleculexe2x80x9d we mean that in a diseased cell, compared to a normal cell, the molecule is present at  greater than 1.2 times the concentration; more preferably  greater than 2 times; still more preferably  greater than 5 times and most preferably  greater than 10 times the concentration. It will be clear that an abnormally elevated amount of a molecule includes the situation where normal (ie wild type) molecules are expressed in cell types where that molecule is not usually expressed (ie presence vs absence). Also, it will be clear that the abnormally elevated amount of a molecule may be due to abnormal activation of expression of a polypeptide which is not normally expressed in a cell or it may be due to an abnormal level of expression.
It is particularly preferred if the CTL administered to the patient is a clonal population of CTL.
It is also particularly preferred if the CTL (preferably a clonal population of CTL) administered to the patient are substantially free of other cell types.
The molecule may be any molecule at least part of which can be presented on the surface of a cell by an HLA class I (or equivalent) molecule.
Preferably, the molecule is a polypeptide including a carbohydrate-containing polypeptide such as a glycoprotein or is a carbohydrate including a peptide-containing carbohydrate, or is a lipid or glycolipid including a peptide-containing lipid or glycolipid.
As discussed in more detail below, abnormal molecules or an abnormally elevated amount of a molecule are associated with many diseases and diseased cells.
The method is particularly advantageous as it is effective in targeting self proteins (for example, those which are overexpressed in the diseased cell or are expressed in a disease cell whereas in a normal cell of the same type they are not expressed).
The patient may or may not be immuno-suppressed when receiving the CTL. It is preferred if the patient is immuno-suppressed.
It is more preferred if the said molecule is a polypeptide. It is well known in the art of immunology that peptide fragments derived from larger peptides or polypeptides are presented by HLA class I (or equivalent) molecules on the surface of a cell, especially diseased cells.
Although the CTL may be derived from the individual who is the patient from a sample taken before the patient acquired the disease, it is most preferred if the CTL are derived from an individual other than the patient.
Of course, it is preferred that the individual is a healthy individual. By xe2x80x9chealthy individualxe2x80x9d we mean that the individual is generally in good health, preferably has a competent immune system and, more preferably, is not suffering from any disease which can be readily tested for, and detected.
In a particularly preferred embodiment the CTL are derived from an individual which individual does not carry the HLA class I (or equivalent) molecule type which, in the patient, presents at least part of said abnormal molecule, or molecule abnormally elevated, contained in or associated with the diseased cells of said patient.
The word xe2x80x9ctypexe2x80x9d is used in the conventional immunological sense.
Thus, the CTL are derived from an individual whose HLA class I (or equivalent) molecules are mismatched with those of the patient. Thus, it is preferred if the CTL are allo-restricted.
In this preferred embodiment the HLA class I (or equivalent) molecule types, other than the type that presents at least part of said abnormal molecule or said molecule abnormally elevated, may be the same or different as between the patient and the individual. In certain circumstances it is preferred if they are the same.
Mutant polypeptides, as are described in more detail below, are often associated with diseased cells and often serve as a molecular marker for the diseased cell. Thus, it is preferred if the polypeptide is a mutant polypeptide associated with said diseased cells.
Diseased cells, as described in more detail below, are often associated with the presence of a polypeptide at a higher level in said diseased cells compared to non-diseased cells. For example, certain polypeptides are known to be overexpressed in some tumour cells. Thus, it is also preferred to target non-mutant self proteins.
It is preferred if the polypeptides are any of the following:
i) normal cellular proteins that are expressed at abnormally high levels in tumours; eg cyclin D1 in a variety of tumours; cyclin E in breast cancer; mdm 2 in a variety of tumours; EGF-R, erb-B2, erb-B3, FGF-R, insulin-like growth factor receptor, Met, myc, p53 and BCL-2 are all expressed in various tumours.
ii) normal cellular proteins that are mutated in tumours; eg Ras mutations in a variety of tumours; p53 mutations in a variety of tumours; BCR/ABL translocation in CML and ALL; CSF-1 receptor mutations in AML and MDS; APC mutations in colon cancer; RET mutations in MEN2A, 2B and FMTC; EGFR mutations in gliomas; PML/RARA translocation in PML; E2APBX1 translocation in pre B leukaemias and in childhood acute leukaemias.
iii) virally encoded proteins in tumours associated with viral infection; eg human papilloma virus proteins in cervical cancer; Epstein-Barr virus proteins in B cell lymphomas and Hodgkin""s lymphoma; HTLV-1 proteins in adult T cell leukaemia; hepatitis B and C virus proteins in hepatocellular carcinoma; herpes-like virus proteins in Kaposi""s sarcoma.
iv) HIV encoded proteins in HIV infected patients.
Thus, the antigens recognised by tumour-reactive CTL can be divided into three main categories: (i) normal self antigens expressed at high levels in tumour cells; (ii) mutated self antigens expressed in tumour cells; (iii) viral antigens expressed in tumours associated with viral infection. Category (i) is preferred.
Three subtypes are included in category (i):
a) normal cellular proteins that are overexpressed;
b) proteins that are expressed in a tissue-specific fashion in normal cells but also in tumours; and
c) proteins that are embryonic antigens, silent in most adult tissues but aberrantly expressed in tumours.
Examples of b) and c) are:
b) tissue-specific differentiation antigens as targets for tumour-reactive CTL such as GATA-1, IKAROS, SCL (expressed in the haematopoietic lineage and in leukaemias); and immunoglobulin constant regions (for treatment of multiple myeloma); and
c) Wilms-tumour antigen 1 (WT1) for treatment of leukaemias and Wilms tumour and carcinoembryonic antigens (CEA a foetal protein) for liver and intestinal tumours.
Overexpression of oncogene-encoded proteins in human tumours and mutated oncogenes expressed in human tumours are described in Stauss and Dahl (1995) Tumour Immunology, Dalgleish/Browning, Chapter 7, incorporated herein by reference.
Thus, it is preferred if the disease to be treated is cancer; more preferably any one of breast cancer; bladder cancer; lung cancer; prostate cancer; thyroid cancer; leukaemias and lymphomas such as CML, ALL, AML, PML; colon cancer; glioma; seminoma; liver cancer; pancreatic cancer; bladder cancer; renal cancer; cervical cancer; testicular cancer; head and neck cancer; ovarian cancer; neuroblastoma and melanoma.
CML is chronic myelocytic leukaemia; ALL is acute lymphoblastic leukaemia; AML is acute myelocytic leukaemia; and PML is pro-myelocytic leukaemia.
The disease to be treated may be any disease caused by a pathogen, particularly a bacterium, yeast, virus, trypanosome and the like. It is preferred if the disease is caused by a chronic infection with a pathogen. It is also preferred if the pathogen is one which is not readily cleared by the host immune system.
It is preferred if the disease is a viral infection; more preferably a disease caused by any one of HIV, papilloma virus, Epstein-Barr virus, HTLV-1, hepatitis B virus, hepatitis C virus, herpes virus or any virus that causes chronic infection. It is particularly preferred if the virus is HIV.
Abnormal glycosylation of polypeptides is also known to occur in some diseases and diseased cells.
Abnormally elevated amounts of a hormone produced by cells occur in some diseases such as certain types of thyroid disease. Thus, the method of the invention is usefully employed to ablate the cells producing the elevated amounts of the hormone. It will be appreciated that, even if the hormone itself, or at least a part thereof, is not presented by an HLA class I (or equivalent) molecule, there may be molecules in the cell which are either abnormal or abnormally elevated and which are presented by an HLA class I (or equivalent) molecule. For example, in certain diseases where a hormone is overproduced by a cell, the biosynthetic enzymes involved in synthesis of said hormone may be overproduced by the cell.
Bacterial infections, particularly those that cause chronic infection may also be usefully treated by-the method of the invention. It is preferred if the bacterial infection is an intracellular infection. Thus, the method may be useful in treating tuberculosis.
The method may also be used to treat malaria.
It is preferred if the HLA class I (or equivalent) molecule type of the patient is determined prior to administration of CTL. This is particularly preferred when the CTL are derived from an individual other than the patient whose HLA class I (or equivalent) molecules are mismatched with those of the patient.
Because of the very extensive study of the genetics of the HLA class I system the type can readily be determined using DNA typing. In particular it is convenient to use a DNA amplification-based typing system such as PCR. These methods are well known in the art and can be employed on a small tissue sample such as a saliva sample or scrape of mouth epithelial cells.
It will be appreciated that the method of the invention may be employed with any mammal such as human, cat, dog, horse, cow, sheep or pig.
It is most preferred if the patient is a human.
Although it is preferred that the patient and the donor of the CTL are the same species, for example both human, it is contemplated that the method is also useful where the patient and the donor are from different species. In other words, the method of the first aspect of the invention includes that a human patient may be given CTL from a non-human donor.
The cytotoxic T lymphocytes for use in the method of the invention, particularly a clonal population of CTL, can conveniently be made using the method of the third aspect of the invention described below.
A particularly preferred embodiment of the first aspect of the invention is wherein the HLA class I (or equivalent) molecule type of the patient is determined prior to administration of the CTL, the CTL are derived from an individual which individual does not carry the HLA class I (or equivalent) molecule type which, in the patient, presents at least part of said abnormal molecule, or molecule abnormally, elevated contained in or associated with the diseased cells of said patient, and the CTL is selected from a library of CTL clones, said library comprising a plurality of CTL clones each derived from an individual with a different HLA class I (or equivalent) molecule type and each said CTL clone recognises said diseased cells.
More preferably each said CTL clone recognises at least part of the same molecule contained in or associated with said diseased cells.
It is preferred if between about 108 and 1011 CTL are administered to the patient; more preferably between 109 and 1010 CTL. The cells may be given to a patient who is being treated for the disease by some other method. Thus, although the method of treatment may be used alone it is desirable to use it as an adjuvant therapy.
The CTL may be administered before, during or after the other therapy.
When the disease to be treated is a cancer it is preferable if the cancer has been, is being or will be treated with a conventional therapy or surgery as well as with the method of the invention. Conveniently, depending on the therapy, the cancer is treated by radiotherapy or by chemotherapy.
When the disease to be treated is an infection by a pathogen it is preferable if the infection has been, is being or will be treated with a conventional therapy or surgery.
If the patient to be treated has HIV infection it is preferable if the method of the invention is used as an adjuvant to treatment with a reverse transcriptase inhibitor such as AZT or 3TC.
When the method of the invention is used to treat a solid tumour it is preferred if the CTL are administered as the first post-surgery treatment.
When the method of the invention is used to treat leukaemia it is preferred if the CTL are administered after radiotherapy or chemotherapy. It is also preferred if leukaemia patients are also treated with the CTL in combination with bone marrow transplantation.
The CTL may be administered by any convenient route. It is preferred if the CTL are administered intravenously. It is also preferred if the CTL are administered locally to the site of the disease (such as a tumour or local viral or bacterial infection). Conveniently, the CTL are administered into an artery that supplies the site of the disease or the tissue where the disease is located.
A second aspect of the invention provides use of cytotoxic T lymphocytes (CTL) in the manufacture of a medicament for treating a patient with a disease wherein the patient contains diseased cells which cells contain, or are associated with, an abnormal molecule or an abnormally elevated amount of a molecule and which cells are capable of presenting at least part of said molecule on their surface by an HLA class I (or equivalent) molecule, wherein the cytotoxic T lymphocytes recognise at least part of said molecule when presented by an HLA class I (or equivalent) molecule on the surface of a cell and they are not derived from the patient with a disease.
A third aspect of the invention provides a method of making a clonal population of cytotoxic T lymphocytes (CTL) reactive against a selected molecule the method comprising the step of (a) co-culturing a sample containing CTL, or a precursor thereof, derived from a healthy individual with a stimulator cell which expresses HLA class I (or equivalent) molecules on its surface and that presents at least a part of the selected molecule in a large proportion of occupied said HLA class I (or equivalent) molecules present on the surface of said stimulator cell and (b) selecting a CTL clone reactive against said selected molecule when at least a part of said molecule is presented by an HLA class I (or equivalent) molecule on the surface of a cell.
It will be appreciated that the stimulator cells of the method may be made using the methods described in WO 93/17095, incorporated herein by reference, and it will be appreciated that the method steps of the method are essentially the same as those described in WO 93/17095 with the very important exception that in the present case the method involves co-culturing a sample containing CTL or a precursor thereof derived from a healthy individual with a stimulator cell whereas the method of WO 93/17095 makes use of a source of CTL from a patient to be treated with the cells. In addition, the present invention, in contrast to WO 93/17095, prefers raising CTL against peptides presented by allogeneic not syngeneic HLA class I (or equivalent) molecules.
In particular, the following-portions of WO 93/17095 are incorporated by reference: the xe2x80x9cDetailed Descriptionxe2x80x9d on pages 23 to page 52, line 11 which describes the production of a stimulator cell; the section on the generation of peptides with optimal binding characteristics for Class I molecules on page 90 onwards; and the Class I molecule bank described on pages 123 and 124.
By xe2x80x9clarge proportionxe2x80x9d we mean at least 50% of the occupied HLA Class I (or equivalent) moleculesxe2x80x9d, more preferably at least 70%, still more preferably at least 90% and most preferably at least 99%xe2x80x9d.
A xe2x80x9csample containing CTL or a precursor thereofxe2x80x9d may be any suitable such sample and specifically includes, but is not limited to, peripheral blood mononuclear cells (PBMC), umbilical cord blood (which is a naive T cell source), any tissue which contains an invasion of T cells and any body fluid which contains T cells or precursors thereof, and includes thymocytes.
The sample containing CTL may or may not be a culture of CTL which have been cloned in vitro.
Preferably, said sample containing CTL or a precursor thereof is PBMC.
Preferably, said molecule is a polypeptide.
Suitably, said selected molecule is an abnormal molecule associated with a diseased cell, or a molecule associated with a diseased cell wherein an abnormally elevated amount of said molecule is present in said diseased cell.
By xe2x80x9cmolecule associated with a diseased cellxe2x80x9d we include any molecule which is found in an abnormal form in the diseased cell or is found in abnormally elevated levels in the diseased cells. Of course, it is most convenient if the said selected molecules, and more particularly the parts thereof presented by the HLA class I (or equivalent) molecules on the stimulator cells, are synthetic equivalents of peptides produced by processing of cellular proteins (which may be intracellular, surface expressed, secreted and so on), and HLA-associated presentation on the cell. Methods are known, particularly computer-based methods using peptide motifs, for selecting a peptide sequence from a larger polypeptide wherein said peptide sequence is a good candidate for binding to a particular HLA class I molecule (or equivalent) type. In particular, it is preferred if said selected molecules are synthesised in vitro. It is particularly preferred if the part of the selected molecule is a peptide and this is made by standard peptide synthetic methods. Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutarine or asparagine are C-terminal residues, use is made of the 4,4xe2x80x2-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide 1-hydroxybenzotiazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesised. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, or by MALDI (matrix assisted laser desorption ionisation) mass spectrometry or electrospray mass spectrometry.
Conveniently, said selected molecule is a mutant polypeptide associated with a diseased cell or a polypeptide present at a higher level in said diseased cell compared to a non-diseased cell.
Preferably said diseased cell is any one of a cancer cell, a virus-infected cell, a bacterium-infected cell and a cell expressing an abnormally elevated amount of a hormone.
More preferably the healthy individual is a human. It is also preferred that the CTL are raised against peptides presented by allogeneic not syngeneic HLA class I (or equivalent) molecules.
It is preferred if the polypeptides are any of the following:
i) normal cellular proteins that are expressed at abnormally high levels in tumours; eg cyclin D1 in a variety of tumours; cyclin E in breast cancer; mdm 2 in a variety of tumours; EGF-R, erb-B2, erb-B3, FGF-R, insulin-like growth factor receptor, Met, myc, p53 and BCL-2 are all expressed in various tumours.
ii) normal cellular proteins that are mutated in tumours; eg Ras mutations in a variety of tumours; p53 mutations in a variety of tumours; BCR/ABL translocation in CML and ALL; CSF-1 receptor mutations in AML and MDS; APC mutations in colon cancer; RET mutations in MEN2A, 2B and FMTC; EGFR mutations in gliomas; PML/RARA translocation in PML; E2A-PBX1 translocation in pre B leukaemias and in childhood acute leukaemias.
iii) virally encoded proteins in tumours associated with viral infection; eg human papilloma virus proteins in cervical cancer; Epstein-Barr virus proteins in B cell lymphomas and Hodgkin""s lymphoma; HTLV-1 proteins in adult T cell leukaemia; hepatitis B and C virus proteins in hepatoceliular carcinoma; herpes-like virus proteins in Kaposi""s sarcoma.
iv) HIV encoded proteins in HIV infected patients.
Three subtypes are included in category (i):
a) normal cellular proteins that are overexpressed;
b) proteins that are expressed in a tissue-specific fashion in normal cells but also in tumours; and
c) proteins that are embryonic antigens, silent in most adult tissues but aberrantly expressed in tumours.
Examples of b) and c) are:
b) tissue-specific differentiation antigens as targets for tumour-reactive CTL such as GATA-1, IKAROS, SCL (expressed in the haematopoietic lineage and in leukaemias); and immunoglobulin constant regions (for treatment of multiple myeloma); and
c) Wilms-tumour antigen 1 (WT1) for treatment of leukaemias and Wilms tumour and carcinoembryonic antigens (CEA a foetal protein) for liver and intestinal tumours.
In a particularly preferred embodiment the method leads to the isolation of CTL clones that recognise peptides presented by HLA class I molecules of cancer patients or HIV patients. The CTL are preferably isolated from HLA mismatched, healthy individuals. In particular, it is preferred if the healthy individual does not carry the HLA class I (or equivalent) molecule type which, on the stimulator cell, presents at least a part of the selected molecule. This will ensure that the CTL repertoire of the healthy responder will not be tolerant to the part of the selected molecule presented by the HLA molecule of the patient. This is because of the fact that T cell tolerance is self-HLA restricted. This means that the CTL of a healthy responder individual will be tolerant only to peptide fragments presented by his/her own HLA molecules, but not to peptide fragments presented by mismatched HLA molecules of a patient. Thus, it is preferred if the CTL which are made using this aspect of the invention are allo-restricted and are allogeneic with respect to the patient. Once isolated, the CTL can be used for adoptive immunotherapy of all patients expressing appropriate HLA class I molecules as described in the method of the first aspect of the invention. Conveniently, the method of this third aspect of the invention is used to generate a bank or library of CTL clones recognising peptides derived from tumour associated proteins or HIV proteins presented by different HLA class I molecules. This bank of CTL clones is available for patients expressing the appropriate HLA class I molecules. Thus, adoptive immunotherapy will no longer depend upon the elaborate production of autologous CTL clones for each patient, but will be achieved with xe2x80x98ready to goxe2x80x99 heterologous CTL clones.
The method of this aspect of the invention is particularly suited for the production of CTL against self proteins that are expressed at abnormally high levels in tumours or against self proteins that are expressed in tumours and in a limited number of normal cells (tissue-specific differentiation antigens), or against embryonic antigens whose expression is activated in tamour cells. It is possible that cancer patients are frequently tolerant to self peptides derived from these proteins and cannot mount CTL responses. This is different in HLA mismatched individuals. Their T cell repertoire will not be tolerant to self peptides presented in the context of the class I molecules expressed by HLA mismatched cancer patients. Therefore, using HLA mismatched, healthy individuals, it will be possible to isolate CTL which recognise self peptides presented by class molecules of cancer patients. By definition, such CTL are molecule-specific, usually peptide-specific, and restricted by allogeneic class I molecules. These CTL are expected to efficiently lyse tumour cells presenting these peptides, whilst normal cells do not present these peptides or the levels of presentation are too low to stimulate CTL lysis.
In addition to abnormally expressed self peptides, mutated self peptides derived from mutated oncogenes, or viral peptides derived from HIV also represent targets for adoptive immunotherapy. Thus, in a further preferred embodiment, CTL are generated in vitro from healthy individuals. These CTL are specific for the mutated or viral peptides presented by HLA class I molecules of cancer patients or HIV infected patients. The peptide presenting class I alleles may be shared between the patients and the healthy donors, in which case the in vitro generated CTL will be self HLA-restricted. Alternatively and preferably, patients and healthy donors may be HLA mismatched, in which case the CTL will be allo-restricted. Allo-restricted CTL may be advantageous in situations where the precursor frequency and/or avidity of self-restricted CTL is low.
The method of this aspect of the invention is suitable for generating allo-restricted or self-restricted CTL clones against selected peptides derived from tumour-associated proteins or HIV proteins. The CTL are conveniently generated in vitro by co-culturing PBMC from healthy individuals with stimulator cells that present a tumour-associated or HIV peptide in a large proportion of MHC class I molecules. This facilitates the isolation of CTL clones specific for a complex of selected peptide plus MHC class I molecule expressed by the stimulator cells. Such CTL clones may be useful for adoptive immunotherapy of all patients who express the MHC class I allele against which the CTL have been raised.
The concept of raising allo-restricted, peptide specific CTL is now discussed.
Although the high ligand density model postulates that allo-reactive CTL recognise allogeneic MHC molecules directly, there is currently no conclusive experimental evidence in its support. In contrast, there is good evidence that allo-reactive CTL clones recognise specific peptides presented in the peptide binding groove of allogeneic MHC molecules (8, 9). Therefore, these CTL clones are molecule-specific, usually peptide-specific, and recognition is restricted by allogeneic class I molecules. Nevertheless, the fine specificity of primary CTL responses induced against allogeneic MHC class I molecules is usually unknown. This is because numerous peptides derived from various cellular proteins are presented in the peptide binding groove of MHC class I molecules. Thus, primary allo-restricted CTL responses are inherently poly-specific and directed against numerous MHC bound peptides of unknown sequence. This has previously made it difficult to induce allo-restricted CTL of desired peptide specificity. In addition to this technical difficulty, the possibility of inducing peptide-specific, allo-restricted CTL previously has not been seriously investigated previously because it violates a fundamental immunological concept. The selection of the T cell repertoire takes place in the thymus where two key events occur (10). During negative selection T cells expressing T cell receptors (TCRs) that recognise with high affinity MHC molecules presenting self peptides are deleted from the repertoire. In contrast, TCRs that recognise MHC/peptide complexes with low affinity are positively selected and released into the periphery as mature T cells. It is believed that as a consequence of positive selection the mature T cells are self MHC-restricted. Thus, mature T cells are thought to efficiently recognise immunogenic peptides only when they are presented by self MHC molecules, but not when they are presented by allogeneic MHC molecules.
Here, it is proposed to employ allo-restricted as well as self-restricted CTL from healthy individuals for adoptive immunotherapy. The CTL recognised peptides may be derived from proteins whose expression is activated in tumours, from proteins that are overexpressed in tumours, from tissue-specific proteins that are expressed in tumours, from mutated proteins, or from viral proteins. In experiments described below, we found that it is possible to isolated peptide-specific, allo-restricted CTL. Some allo-restricted CTL clones can recognise very low concentrations of peptides (femtomolar concentrations) indicating that they are at least as sensitive (perhaps even more sensitive) than self-restricted CTL which typically require picomolar peptide concentrations for recognition. We also found that these CTL can be injected three times into immunocompetent hosts without causing any immunological reactions (eg anaphylaxis or hypersensitivity). The allo-restricted CTL clones are probably most efficient for short term treatment of immunocompromised patients. It is unlikely that these CTL will have any long term side effects because they will be eventually eliminated by a functional host immune response.
Allo-restricted CTL may be particularly useful in the treatment of leukaemia. Leukaemia patients, in particular CML patients, are frequently treated by bone marrow transplantation, and there is strong evidence that the disease prognosis is improved when donor CTL can mount an immune response against recipient""s leukaemia cells. It is known that donor CTL in bone marrow transplant recipients can mount an immune response against recipient""s MHC molecules, leading to the clinical picture of graft versus host disease (GvH). In leukaemia patients a low level of GvH is clinically favourable, since it is correlated with prolonged leukaemia free survival (5). This graft versus leukaemia (GvL) effect is most likely due to donor CTL that can recognise and kill recipients leukaemic cells (6, 7). Whether allo-reactive CTL that mediate GvH and GvL are the same or represent distinct CTL populations has remained a controversial issue. This is because the peptide-specificity of CTL involved in GvH and GvL is generally unknown.
The protocol described here can lead to the isolation of CTL clones which mediate GvL without causing GvH. CTL with specificity for leukaemias can be generated against peptides which are expressed in leukaemic cells but not in cells outside the haematopoietic lineage. Such CTL clones can be used for adoptive immunotherapy of leukaemia patients, where they will eliminate leukaemic cells and perhaps also some normal bone-marrow derived cells. The possible loss of normal bone-marrow cells is not expected to cause any problems because these patients are frequently treated with bone marrow transplantation from healthy donors. The following proteins are some of the targets for anti-leukaemia CTL clones: GATA-1, IKAROS, SCL, WT1. GATA-1 and IKAROS are zinc finger-containing DNA binding proteins expressed only in haematopoietic cells. SCL is a helix-loop-helix transcription factor expressed in haematopoietic cells but also in endothelial cells and brain. Wilms tumour 1 (WT1) protein is an embryonic differentiation antigen not normally expressed in adult tissues except for acute and chronic leukaemias.
Except for SCL, these proteins are expressed in leukaemia progenitor cells but not in cells outside the haematopoietic lineage in adults. Peptides derived from these proteins which are presented by HLA-class I molecules are used to raise CTL from donors who express mismatched HLA class I alleles (to circumvent CTL tolerance), or from donors who express matched class I alleles (in case tolerance is not a problem). CTL clones are isolated and their specificity is analysed against leukaemic cells and non-leukaemic control in vitro. Clones with appropriate specificity are used for treatment of all leukaemia patients expressing the HLA class I allele that is the CTL restriction element.
Similarly, allo-restricted CTL clones are believed to be useful for treatment of patients with multiple myeloma. Suitable targets for multiple myeloma-specific CTL include the constant regions of the immunoglobulin heavy and light chain. Peptides are selected from the heavy and light chain constant regions which bind to HLA class I molecules. CTL against these peptides are isolated from HLA mismatched donors in order to circumvent CTL tolerance. These allo-restricted CTL lyse myeloma cells but also normal B cells in patients treated by adoptive immunotherapy. The eliminated B cells will be replaced by new B cells developing in the patient""s bone marrow, whilst elimination of myeloma cells may be permanent.
A particularly preferred embodiment is the generation of allo-restricted CTL against known epitopes in HIV proteins and in tumour-associated proteins. A number of CTL recognised peptides have been identified in various HIV proteins and in tumour-associated proteins. In particular, CTL epitopes have been identified in the HIV env, gag, pol, vif and nef proteins (12, 13). Also, CTL epitopes have been identified in the tumour-associated melanoma proteins tyrosinase, mart1/melanA, gp100/pmel17, mage and bage (14-21). The use of peptides corresponding to these CTL epitopes has the advantage that they are known to be produced by natural antigen processing. CTL produced in this way recognise target cells expressing the relevant proteins endogenously. The exploitation of known CTL epitopes represents a considerable shortcut because it avoids screening of large numbers of test peptides and identification of naturally produced peptides. However, known peptides may represent immunodominant peptides. The method of the third aspect of the invention may be used to identify new peptides, which new peptides may be preferred as they are likely to be subdominant peptides. Since subdominant peptides are less likely to be immunoselected by patient""s CTL responses, they may represent better targets for adoptive immunotherapy. Nevertheless, peptides representing known CTL epitopes can be ideally exploited to generate allo-restricted or self-restricted CTL in vitro and to test their anti-viral and anti-tumour effects in vivo.
The method of the third aspect of the invention allows the isolation of HLA class I-restricted CTL clones specific for peptides produced in tumour cells and for peptides produced in HIV infected cells. Conveniently, SCID mouse models are used to determine the in vivo antitumour and anti-HIV effects of these CTL. These CTL clones are useful for adoptive immunotherapy, especially in humans.
It is preferred if the method of the third aspect of the invention further comprises determining the HLA class I (or equivalent) molecule type of the healthy individual. Conveniently, this is done by DNA analysis as disclosed above.
It is particularly preferred if the stimulator cell has a type of HLA class I (or equivalent) molecule on its surface which HLA class I (or equivalent) molecule type is not present in the healthy individual.
It is particularly preferred if said stimulator cell is a cell which is substantially incapable of itself loading said HLA class I (or equivalent) molecule with at least a part of said selected molecule. As is described in more detail below, the HLA class I (or equivalent) molecule may readily be loaded with at least a part of said selected molecule in vitro.
Conveniently, said cell is a mammalian cell defective in the expression of a peptide transporter such that, when at least part of said selected molecule is a peptide, it is not loaded into said HLA class I (or equivalent) molecule.
Preferably the mammalian cell lacks or has a reduced level or has reduced function of the TAP peptide transporter. Suitable cells which lack the TAP peptide transporter include T2, RMA-S and Drosophila cells. TAP is the Transporter Associated with antigen Processing.
Thus, conveniently the cell is an insect cell such as a Drosophila cell.
The human peptide loading deficient cell line T2 is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA under Catalogue No CRL 1992; the Drosophila cell line Schneider line 2 is available from the ATCC under Catalogue No CRL 19863; the mouse RMA-S cell line is described in Karre and Ljunggren (1985) J. Exp. Med. 162, 1745, incorporated herein by reference.
In a preferred embodiment the stimulator cell is a host cell (such as a T2, RMA-S or Drosophila cell) transfected with a nucleic acid molecule capable of expressing said HLA class I (or equivalent) molecule. Although T2 and RMA-S cells do express before transfection HLA class I molecules they are not loaded with a peptide.
Mammalian cells can be transfected by methods well known in the art. Drosophila cells can be transfected, as described in Jackson et al (1992) Proc. Natl. Acad. Sci. USA 89, 12117, incorporated herein by reference.
Conveniently said host cell before transfection expresses substantially no HLA class I (or equivalent) molecules.
It is also preferred if the stimulator cell expresses a molecule important for T cell costimulation such as any of B7.1, B7.2, ICAM-1 and LEA 3.
The nucleic acid sequences of numerous HLA class I (and equivalent) molecules, and of the costimulator molecules, are publicly available from the GenBank and EMBL databases.
It is particularly preferred if substantially all said HLA class I (or equivalent) molecules expressed in the surface of said stimulator cell are of the same type.
HLA class I in humans, and equivalent systems in other animals, are genetically very complex. For example, there are at least 110 alleles of the HLA-B locus and at least 90 alleles of the HLA-A locus. Although any HLA class I (or equivalent) molecule is useful in this aspect of the invention, it is preferred if the stimulator cell presents at least part of the selected molecule in an HLA class I molecule which occurs at a reasonably high frequency in the human population. It is well known that the frequency of HLA class I alleles varies between different ethnic groupings such as Caucasian, African, Chinese and so on. At least as far as the Caucasian population is concerned it is preferred that HLA class I molecule is encoded by an HLA-0201 allele, or an HLA-A1 allele or an HLA-A3 allele or an HLA-B7 allele. HLA-A0201 is particularly preferred.
When the method of the third aspect of the invention is used to make a library of CTL it is convenient if the HLA alleles which restrict recognition by those CTL clones are selected on the basis of frequency in a particular ethnic grouping.
It will be appreciated that a stimulator cell which expresses HLA class I (or equivalent) molecules on its surface and that presents at least a part of a selected molecule in a large proportion of occupied said HLA class I (or equivalent) molecules present on the surface of said stimulator cell forms a further aspect of the invention.
Preferably the selected molecule is an abnormal molecule or a molecule whose amount is abnormally elevated.
A fourth aspect of the invention provides a clonal population of cytotoxic T lymphocytes reactive against a selected molecule obtainable by the method of the third aspect of the invention.
A fifth aspect of the invention provides a clonal population of cytotoxic T lymphocytes reactive against a selected molecule wherein the said CTL has a high avidity for a cell.
It will be appreciated that, at least for self molecules abnormally elevated, and in particular for self polypeptides expressed at high levels, the method of the third aspect of the invention allows the production of CTL of much higher avidity and sensitivity than can otherwise be produced. This is particularly the case when the stimulator cell has a type of HLA class I (or equivalent) molecule on its surface which HLA class I (or equivalent) molecule type is not present in the healthy individual.
Thus, the method of the third aspect of the invention is preferably used to produce cytotoxic T lymphocytes (CTL) from healthy individuals that can be used for adoptive immunotherapy of cancer patients and patients infected with the human immunodeficiency virus. The CTL are generated entirely in vitro and may be administered to patients intravenously. Since this form of adoptive immunotherapy does not depend upon a functional host immune system, it is believed to be particularly suited to patients who are immunosuppressed, for example as a consequence of HIV infection or radiotherapy and chemotherapy in the case of cancer. Preferably, all peptide-specific CTL are isolated from healthy donors, and no blood samples from patients are required.
The hallmark of the allo-restricted CTL clones described herein is that they can recognise peptides derived from normal cellular proteins presented on the cell surface by MHC class I molecules. Most importantly, the MHC genotype of the allo-restricted CTL clones and of the recognised target cells (or other cells) in the patient is different. The genetic difference does not only apply to the MHC region of the genome, but also to other polymorphic genes. Thus, it is possible that there might be a polymorphism in the gene segments of the TCRxcex1 and xcex2 locus of the CTL clone and the target cell. However, the TCR genes in these cells may be identical even if CTL and target cell are of different genetic origin.
A sixth aspect of the invention provides a clonal population of cytotoxic T lymphocytes according to the fourth or fifth aspects of the invention for use in medicine.
A seventh aspect of the invention provides a pharmaceutical composition comprising clonal population of cytotoxic T lymphocytes according to the fourth or fifth aspects of the invention and a pharmaceutically acceptable carrier.
The aforementioned CTL of the invention or a formulation thereof may be administered by any conventional method including by parenteral (eg subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.
Whilst it is possible for the CTL of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.
An eighth aspect of the invention provides use of a clonal population of cytotoxic T lymphocytes derived from a healthy individual and reactive against a selected abnormal molecule derived from a diseased cell from a patient with a disease, or a selected molecule derived from a diseased cell from a patient with a disease wherein an abnormally elevated amount of said molecule is present in said diseased cell, in the manufacture of a medicament for treating a patient with the disease wherein said healthy individual has a different HLA type to said patient.
A ninth aspect of the invention provides a library of CTL clones, said library comprising a plurality of CTL clones derived from individuals and each said CTL clone is restricted by a different HLA class I allele and recognises a molecule associated with a selected disease.
The library is conveniently stored in a form where each CTL clone retains viability. Conveniently the library is stored frozen.
Preferably, the library contains a selection of CTLs which have been made by the method of the third aspect of the invention. The library may be disease or disease cell specific or it may be HLA class I (or equivalent) molecule type specific. Preferred diseases or HLA class I (or equivalent) molecule types are described above.
Advantageously the library contains CTL for different diseases and/or CTL for different molecules (eg peptides) for the same disease and clones of each of the CTL are restricted by different HLA class I alleles. For an individual patient an appropriate CTL clone is selected by reference to an appropriate peptide (ie one that is presented on their disease cells), and by reference to the HLA class I allele of the CTL such that the CTL bears an HLA class I allele different from that of the patient.
A tenth aspect of the invention provides a therapeutic system comprising (a) means to determine the HLA class I (or equivalent) type of a patient to be treated and (b) a library of CTL clones, said library comprising a plurality of CTL clones derived from individuals with differing HLA class I (or equivalent) molecule type and each said CTL clone recognises a molecule associated with a selected disease.
The method of treating a patient according to a particular embodiment of the first aspect of the invention makes use of allogeneic CTL which are particularly suited for use in adoptive immunotherapy when the antigen recognised is a self-antigen.
However, because of their allogeneic nature, the recipients (patients) are expected to mount immune responses against the transferred CTL in some circumstances, which may limit their half life and their anti-tumour activity in the recipient host (patient). However, immunosuppression of the recipient (patient) is one way of diminishing such host immune responses and the method of the first aspect of the invention is useful.
The other possibility described here is to use autologous CTL which are non-immunogenic when transferred back into the original host. These autologous CTL are manipulated in vitro to express the TCRs isolated from allo-restricted CTL clones.
An eleventh aspect of the invention provides a method of making a cytotoxic T lymphocyte (CTL) suitable for treating a patient, the method comprising (a) making a clonal population of CTL by the method of the third aspect of the invention; (b) preparing a genetic construct capable of expressing the T-cell receptor (TCR) of the said clonal population of CTL, or a functionally equivalent molecule; and (c) introducing said genetic construct into a CTL or precursor thereof which CTL or precursor is derived from said patient.
All of the preferred embodiments of the third aspect of the invention are preferred in this aspect of the invention when making a clonal population of CTL in step (a) of this method. In particular, the CTL isolated in step (a) are preferably isolated from HLA mismatched, healthy individuals (compared to the patient to be treated). Thus, it is particularly preferred if the CTL isolated in step (a) are allo-restricted and are allogeneic with respect to the patient to be treated.
Allogenicity is the situation of two or more different allelic forms of the same protein in different individuals of the same species.
By a xe2x80x9cmolecule functionally equivalent to a T-cell receptorxe2x80x9d we mean any molecule which can perform the same function as a T-cell receptor. In particular, such molecules include genetically engineered three-domain single-chain T-cell receptors as made by the method described by Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658, incorporated herein by reference.
By a xe2x80x9cgenetic construct capable of expressing the T-cell receptor or functionally equivalent moleculexe2x80x9d we include any genetic construct, whether RNA or DNA, which, when inserted into the CTL derived from the patient or a precursor of said cell, can express the T-ceil receptor or functionally equivalent molecule. Any suitable vector may be used such as a plasmid or virus, including retrovirus.
The genetic construct may be made using methods well known in the art such as those described in Sambrook et al (1989) xe2x80x9cMolecular cloning, a laboratory manualxe2x80x9d, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference.
The DNA encoding the TCR or functionally equivalent molecule may be joined to a wide variety of other DNA sequences for introduction into an appropriate host (which may be the CTL derived from the patient, or a precursor thereof, or another host cell). The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
Generally, the DNA is inserted into an expression vector, such as a plasmid or virus or retrovirus, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through known techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to cotransform the desired host cell. However, in the case of introducing the genetic construct into the CTL of the patient, or a precursor thereof, it is preferred if at least 50% of the CTL are transformed or transfected with the genetic construct. More preferably, at least 70% are so transformed or transfected and still more preferably at least 90% or at least 95%.
A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3xe2x80x2-single-stranded termini with their 3xe2x80x2-5xe2x80x2-exonucleolytic activities, and fill in recessed 3xe2x80x2-ends with their polymerizing activities.
The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
A desirable way to modify the DNA is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the-amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
A particularly preferred method is now described.
The TCRs of allo-restricted CTL clones specific for self peptides presented at elevated levels in tumours are cloned. The TCR usage in allo-restricted CTL clones is determined using (i) TCR variable region-specific monoclonal antibodies and (ii) RT-PCR with primers specific for Vxcex1 and Vxcex2 gene families. A cDNA library is prepared from poly-A mRNA extracted from allo restricted CTL clones. Primers specific for the C-terminal portion of the TCR xcex1 and xcex2 chains and for the N-terminal portion of the identified Vxcex1 and xcex2 segments are used. The complete cDNA for the TCR xcex1 and xcex2 chain is amplified with a high fidelity DNA polymerase and the amplified products cloned into a suitable cloning vector. The cloned xcex1 and xcex2 chain genes are assembled into a single chain TCR by the method as described by Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. In this single chain construct the Vxcex1J segment is followed by the Vxcex2DJ segment, followed by the Cxcex2 segment followed by the transmembrane and cytoplasmic segment of the CD3 xcex6 chain. This single chain TCR is then inserted into a retroviral expression vector (a panel of vectors may be used based on their ability to infect mature human CD8+ T lymphocytes and to mediate gene expression: the retroviral vector system Kat is one preferred possibility (see Finer et al (1994) Blood 83, 43). High titre amphotrophic retrovirus are used to infect purified CD8+ T lymphocytes isolated from the peripheral blood of tumour patients following a protocol published by Roberts et al (1994) Blood 84, 2878-2889, incorporated herein by reference. Anti-CD3 antibodies are used to trigger proliferation of purified CD8+ T cells, which facilitates retroviral integration and stable expression of single chain TCRs. The efficiency of retroviral transduction is determined by staining of infected CD8+ T cells with antibodies specific for the single chain TCR. In vitro analysis of transduced CD8+ T cells establishes that they display the same tumour-specific killing as seen with the allo-restricted CTL clone from which the TCR chains were originally cloned. Populations of transduced CD8+ T cells with the expected specificity will be used for adoptive immunotherapy of the tumour patients. Patients will be treated with in between 108 to 1011 (most likely 109-1010) autologous, transduced CTL.
Other suitable systems for introducing genes into CTL are described in Moritz et al (1994) Proc. Natl. Acad. Sci. USA 91, 4318-4322, incorporated herein by reference. Eshhar et al (1993) Proc. Natl. Acad. Sci. USA 90, 720-724 and Hwu et al (1993) J. Exp. Med. 178, 361-366 also describe the transfection of CTL.
Thus, a twelfth aspect of the invention provides a cytotoxic T lymphocyte suitable for treating a patient obtainable by the method of the eleventh aspect of the invention.
A thirteenth aspect of the invention provides a method of treating a patient with a disease wherein the patient contains diseased cells which cells contain, or are associated with, an abnormal molecule or an abnormally elevated amount of a molecule and which cells are capable of presenting at least part of said molecule on their surface by an HLA class I (or equivalent) molecule, the method comprising administering to the patient a therapeutically effective amount of cytotoxic T lymphocytes (CTL) which recognise at least part of said molecule when presented by an HLA class I (or equivalent) molecule on the surface of a cell wherein the CTL is a CTL according to the twelfth aspect of the invention.
A fourteenth aspect of the invention provides the use of cytotoxic T lymphocytes in the manufacture of a medicament for treating a patient with a disease wherein the patient contains diseased cells which cells contain, or are associated with, an abnormal molecule or an abnormally elevated amount of a molecule and are capable of presenting at least part of said molecule on their surface by an HLA class I (or equivalent) molecule, wherein the cytotoxic T lymphocytes recognise at least part of said molecule when presented by an HLA class I (or equivalent) molecule on the surface of a cell and wherein the CTL is a CTL according to the twelfth aspect of the invention.
The preferred methods of administration, the preferred diseases, and the preferred amounts of CTL administered to treat are the same for the thirteenth aspect of the invention as for the first aspect of the invention.
It will be appreciated that a genetic construct and a library of genetic constructs may be prepared, each capable of expressing a specific TCR, or a functionally equivalent molecule, by making clonal populations of CTL by the method of the third aspect of the invention and preparing a genetic construct capable of expressing the T-cell receptor of the said clonal population of CTL, or a functionally equivalent molecule as described above.
It is particularly convenient if each genetic construct represents a TCR (by way of a TCR or a functionally equivalent molecule) which corresponds to the TCR from a particular CTL from a healthy individual of a known HLA genotype and which CTL was produced by co-culturing with a stimulator cell which expresses a known HLA class I (or equivalent) molecule on its surface which HLA class I or equivalent molecule binds at least a part of a given molecule on its surface.
In this way it is possible to generate libraries of genetic constructs each construct of which can be introduced into a patient""s CTL or precursor and which genetic construct capable of expressing a TCR or functionally equivalent molecule can be selected on the basis of the patient""s HLA genotype and disease to be treated. The particular genetic construct representing a TCR is selected on the basis of the specificity of the T cell clone (ie according to what is expressed by the patient""s disease cell, especially a tumour cell).
Preferably, the HLA genotype of the patient and the HLA genotype of the CTL from which the TCR or functionally equivalent molecule expressed by the genetic construct is derived are mismatched.
In general, it is easy to define a TCR as allo-restricted as long as it is expressed in the original CTL clone. However, once the TCR genes are isolated and transferred into patients CTL, it becomes very difficult to define them as allo-restricted. A sequence comparison between the sequence of the transferred TCR and the endogenous TCR genes can identify them as a non-self TCR genes in those cases where there is a polymorphism in the TCR genes. Non-self TCRs are, however, not necessarily derived from allo-restricted CTL clones.
The invention will now be described in more detail with reference to the following Examples and Figures wherein: